From 2010.igem.org

We used gel electrophoresis to confirm the quality of our DNA. As we already know, the region our primers amplify is about 300bps. We used 100 bp DNA ladder as a guidance for our bands. The two bands produced were from two separate chromosomal preps.

Lane 1: 100 bp DNA ladder
Lane 2: Chromosomal DNA with ara gene 1
Lane 3: Chromosomal DNA with ara gene 2

Conclusion

The aim of this whole experiment was to extract genomic DNA from Bacillus subtilis ATCC 6633. In order to test whether we had extracted the DNA, we first used PCR to amplify and then used Gel Electrophoresis to compare the bands of ara genes to the 100 bp ladder. It worked!

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 {{Team:Newcastle/mainbanner}}
-=Chromosomal prep=
+=PCR to test chromosomal prep=
 ==Aim==
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 ==Materials and Procedure==
 Please refer to:[[Team:Newcastle/PCR| PCR]] and [[Team:Newcastle/Gel electrophoresis| Gel electrophoresis]] for materials and protocol.
-We used ''ara'' and ''sac'' forward and reverse primers for amplification.
+We used ''ara'' forward and reverse primers for amplification.
 ==Results==
-We used Gel Electrophoresis to test whether we have the DNA we wanted. As we already know, the ''ara'' gene is about 300bps. We used 100 bp DNA ladder as a guidance for our bands. The two bands produced were from two separate ''ara'' cultures and they indeed show the bands in the same region, i.e. 300bps.
+We used gel electrophoresis to confirm the quality of our DNA. As we already know, the region our primers amplify is about 300bps. We used 100 bp DNA ladder as a guidance for our bands. The two bands produced were from two separate chromosomal preps.
 [[Image:Newcastle_Genomic_extraction.jpg|400px]]
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 The aim of this whole experiment was to extract genomic DNA from ''Bacillus subtilis'' ATCC 6633. In order to test whether we had extracted the DNA, we first used PCR to amplify and then used Gel Electrophoresis to compare the bands of ''ara'' genes to the 100 bp ladder. It worked!
-=LacI BioBrick Construction=
-==Aims==
-* To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
-==Materials==
-* Digested plasmids pMutin4 and pSB1AT3
-* PCR product
-* Restriction enzymes EcoR1 and Spe1
-==Protocol==
-* The digests and PCR product are run on an [[Team:Newcastle/Gel_electrophoresis|agarose gel]]
-* pSB1AT3 is excised from the gel
-* ''lacI'' PCR product is purified.
-* Purified ''lacI'' is [[Team:Newcastle/Restriction_digests|restriction digested]] with EcoR1 and Spe1.
-==Results==
-* Bands were seen at the correct size for pMutin4 and pSB1AT3 and the amplified PCR product.
-N.B. To limit DNA exposure to UV radiation images were not taken using the geldoc and bands were extracted directly using UV transilluminator.
-==Conclusion==
-* The digested plasmids and the PCR product were run on an agarose gel in order to determine what was in these samples. The samples should have run at                 . The samples did in fact run in this manner, and so it can be said with confidence that our protocols have worked as they should.
-==Inference==
-* The purified PCR product is cut to give it sticky ends complementary to the digested pSB1AT3. pSB1AT3 is excised from the gel ready for gel extraction.
 {{Team:Newcastle/footer}}

Team:Newcastle/7 July 2010

From 2010.igem.org

Latest revision as of 15:09, 27 October 2010

Contents

PCR to test chromosomal prep

Aim

Materials and Procedure

Results

Conclusion